scholarly journals In vitro transcription profiling of the σ S subunit of bacterial RNA polymerase: re-definition of the σ S regulon and identification of σ S -specific promoter sequence elements

2011 ◽  
Vol 39 (13) ◽  
pp. 5338-5355 ◽  
Author(s):  
Anna Maciąg ◽  
Clelia Peano ◽  
Alessandro Pietrelli ◽  
Thomas Egli ◽  
Gianluca De Bellis ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Promoter Sequence ◽  
In Vitro Transcription ◽  
Transcription Profiling ◽  
Bacterial Rna ◽  
Sequence Elements ◽  
Definition Of

scholarly journals Genome-wide effects onEscherichia colitranscription from ppGpp binding to its two sites on RNA polymerase

2019 ◽  
Vol 116 (17) ◽  
pp. 8310-8319 ◽  
Author(s):  
Patricia Sanchez-Vazquez ◽  
Colin N. Dewey ◽  
Nicole Kitten ◽  
Wilma Ross ◽  
Richard L. Gourse
Keyword(s):  
Rna Polymerase ◽  
Dna Sequences ◽  
Binding Sites ◽  
Promoter Sequence ◽  
In Vitro Transcription ◽  
Transcriptional Responses ◽  
Transcription Analysis ◽  
Genome Wide

The second messenger nucleotide ppGpp dramatically alters gene expression in bacteria to adjust cellular metabolism to nutrient availability. ppGpp binds to two sites on RNA polymerase (RNAP) inEscherichia coli, but it has also been reported to bind to many other proteins. To determine the role of the RNAP binding sites in the genome-wide effects of ppGpp on transcription, we used RNA-seq to analyze transcripts produced in response to elevated ppGpp levels in strains with/without the ppGpp binding sites on RNAP. We examined RNAs rapidly after ppGpp production without an accompanying nutrient starvation. This procedure enriched for direct effects of ppGpp on RNAP rather than for indirect effects on transcription resulting from starvation-induced changes in metabolism or on secondary events from the initial effects on RNAP. The transcriptional responses of all 757 genes identified after 5 minutes of ppGpp induction depended on ppGpp binding to RNAP. Most (>75%) were not reported in earlier studies. The regulated transcripts encode products involved not only in translation but also in many other cellular processes. In vitro transcription analysis of more than 100 promoters from the in vivo dataset identified a large collection of directly regulated promoters, unambiguously demonstrated that most effects of ppGpp on transcription in vivo were direct, and allowed comparison of DNA sequences from inhibited, activated, and unaffected promoter classes. Our analysis greatly expands our understanding of the breadth of the stringent response and suggests promoter sequence features that contribute to the specific effects of ppGpp.

scholarly journals Regulation of Gene Expression of phiEco32-like Bacteriophage 7-11

2022 ◽  
Author(s):  
Daria Lavysh ◽  
Vladimir Mekler ◽  
Evgeny Klimuk ◽  
Konstantin Severinov
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Regulation Of Gene Expression ◽  
In Vitro Transcription ◽  
Single Element ◽  
Bacterial Rna ◽  
Sigma 70 ◽  
Rna Polymerase Holoenzyme ◽  
Rna Polymerase Promoter

Salmonella enterica serovar Newport bacteriophage 7-11 shares 41 homologous ORFs with Escherichia coli phage phiEco32 and both phages encode a protein similar to bacterial RNA polymerase promoter specificity  subunit. Here, we investigated the temporal pattern of 7-11 gene expression during the infection and compared it to the previously determined transcription strategy of phiEco32. Using primer extension and in vitro transcription assays we identified eight promoters recognized by host RNA polymerase holoenzyme containing 7-11  subunit SaPh711_gp47. These promoters are characterized by a bipartite consensus GTAAtg-(16)-aCTA and are located upstream of late phage genes. While dissimilar from single-element middle and late promoters of phiEco32 recognized by holoenzyme formed by the phi32_gp36  factor, the 7-11 late promoters are located at genome positions similar to those of phiEco32 middle and late promoters. Two early 7-11 promoters are recognized by RNA polymerase holoenzyme containing host primary σ70 factor. Unlike the case of phiEco32, no shut off of σ70-dependent transcription is observed during 7-11 infection and there are no middle promoters. These differences can be explained by the fact that phage 7-11 does not encode a homologue of phi32_gp79, an inhibitor of host and early phage transcription and an activator of transcription by the phi32_gp36-holoenzyme.

scholarly journals Identification of Promoters Recognized by RNA Polymerase-σE Holoenzyme from Thermus thermophilus HB8

2007 ◽  
Vol 189 (23) ◽  
pp. 8758-8764 ◽  
Author(s):  
Akeo Shinkai ◽  
Naomi Ohbayashi ◽  
Takaho Terada ◽  
Mikako Shirouzu ◽  
Seiki Kuramitsu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcriptional Activity ◽  
Thermus Thermophilus ◽  
Promoter Sequence ◽  
In Vitro Transcription ◽  
Thermophilic Bacterium ◽  
Thermus Thermophilus Hb8 ◽  
Core Enzyme ◽  
Σ Factor

ABSTRACT Thermus thermophilus σE, an extracytoplasmic function σ factor from the extremely thermophilic bacterium Thermus thermophilus HB8, bound to the RNA polymerase core enzyme and showed transcriptional activity. With the combination of in vitro transcription assay and GeneChip technology, we identified three promoters recognized by σE. The predicted consensus promoter sequence for σE is 5′-CA(A/T)(A/C)C(A/C)-N15-CCGTA-3′.

scholarly journals Definition of the Binding Architecture to a Target Promoter of HP1043, the Essential Master Regulator of Helicobacter pylori

2021 ◽  
Vol 22 (15) ◽  
pp. 7848
Author(s):  
Annamaria Zannoni ◽  
Simone Pelliciari ◽  
Francesco Musiani ◽  
Federica Chiappori ◽  
Davide Roncarati ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Response Regulator ◽  
Consensus Sequence ◽  
Binding Mode ◽  
In Vitro Transcription ◽  
Target Sequence ◽  
Computational Alanine Scanning ◽  
Definition Of ◽  
Target Promoter

HP1043 is an essential orphan response regulator of Helicobacter pylori orchestrating multiple crucial cellular processes. Classified as a member of the OmpR/PhoB family of two-component systems, HP1043 exhibits a highly degenerate receiver domain and evolved to function independently of phosphorylation. Here, we investigated the HP1043 binding mode to a target sequence in the hp1227 promoter (Php1227). Scanning mutagenesis of HP1043 DNA-binding domain and consensus sequence led to the identification of residues relevant for the interaction of the protein with a target DNA. These determinants were used as restraints to guide a data-driven protein-DNA docking. Results suggested that, differently from most other response regulators of the same family, HP1043 binds in a head-to-head conformation to the Php1227 target promoter. HP1043 interacts with DNA largely through charged residues and contacts with both major and minor grooves of the DNA are required for a stable binding. Computational alanine scanning on molecular dynamics trajectory was performed to corroborate our findings. Additionally, in vitro transcription assays confirmed that HP1043 positively stimulates the activity of RNA polymerase.

scholarly journals Bacteriophage SP6-specific RNA polymerase. II. Mapping of SP6 DNA and selective in vitro transcription.

1982 ◽  
Vol 257 (10) ◽  
pp. 5779-5788 ◽  
Author(s):  
G A Kassavetis ◽  
E T Butler ◽  
D Roulland ◽  
M J Chamberlin
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
In Vitro Transcription
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